Materials and methods for suppressing and/or treating bone related diseases and symptoms

ABSTRACT

Various aspects and embodiments disclosed herein relate generally to the modelling, treatment, reducing resistence to the treatment, prevention, and diagnosis of diseases/symptoms induced by multiple myeloma. Embodiments include methods of treating a bone related disease, comprising the steps of: administering to a subject at least one therapeutically effective dose of a compound disclosed herein.

PRIORITY CLAIM

This application claims the benefit of U.S. Provisional Pat. ApplicationNo. 62/540,396, filed Aug. 2, 2017, the disclosure of which isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

Various aspects and embodiments disclosed herein relate generally to themodeling, treatment, reducing resistance to the treatment, prevention,and diagnosis of diseases/symptoms induced by multiple myeloma and/orother bone related diseases.

STATEMENT OF GOVERNMENTAL RIGHTS

This invention was made with government support under DK076007 andCA209882 awarded by National Institutes of Health and BX002104 meritaward by the Veterans Administration. The government has certain rightsin the invention.

BACKGROUND

Multiple myeloma is a plasma cell malignancy characterized by expansionof monoclonal plasma cells in the bone marrow (BM) and the presence ofosteolytic lesions. Multiple myeloma has one of the highest incidencesof bone involvement among malignant diseases. It is estimated that up to90% of patients with multiple myeloma have evidence of osteolysis in theform of generalized osteopenia or discrete lytic lesions, and up to 60%of multiple myeloma patients develop pathologic fractures. Multiplemyeloma patients present with severe bone pain caused by osteolyticlesions that rarely heal. The osteolytic lesions are thought to resultfrom increased bone resorption and concomitant long-term suppression ofbone formation. The bone and BM microenvironment is a major contributorto tumor growth and bone destructive process in multiple myeloma.

Notch signaling mediates cell-to-cell communication among myeloma cellsand other cells in the bone marrow favoring growth and survival ofmyeloma cells and increasing osteoclast formation. In vitro and in vivostudies demonstrated that systemic inhibition of Notch signaling usinggamma-secretase inhibitors (GSIs) decreases the growth of myeloma cellsand osteoclast differentiation. However, the use of GSIs in the clinicis limited by the presence of severe adverse side effects such asfatigue, skin disorders, and acute gastrointestinal toxicity. Therefore,development of a new class of drugs is much needed.

SUMMARY OF THE INVENTION

A first embodiment includes at least one compound of the formula A-Y-B,or a pharmaceutically acceptable salt thereof, or a metabolite thereof,wherein A is at least one agent that reduces and/or inhibits theactivitiy of gamma-secretase; B is at least one bone-targeting molecule;and Y is a linker that joins and/or links A and B.

A second embodiment includes the compound according to the firstembodiment, wherein A is the at least one agent that reduces and/orinhibits the activitiy of gamma-secretase; Y is the linker comprisingthe formula NR₁;

-   R₁ is NR₂R₃, NR₂S(=O)₂R₃ or R₂OR₃;-   R₂ and R₃ are independently selected from H; C₁-C₃ alkyl, C₁-C₄    alkyl, C₁-C₅ alkyl, C₁-C₆ alkyl, C₁-C₇ alkyl, C₁-C₈ alkyl, C₁-C₉    alkyl, C₁-C₁₀ alkyl, C₁ alkyl, C₂ alkyl, C₃ alkyl, C₄ alkyl, C₅    alkyl, C₅ alkyl, C₆ alkyl, C₇ alkyl, C₈ alkyl, C₉ alkyl, C₁₀ alkyl,    or any combination thereof; C₁-C₃ alkoxy, C₁-C₄ alkoxy, C₁-C₅    alkoxy, C₁-C₆ alkoxy, C₁-C₇ alkoxy, C₁-C₈ alkoxy, C₁-C₉ alkoxy,    C₁-C₁₀ alkoxy, C₁ alkoxy, C₂ alkoxy, C₃ alkoxy, C₄ alkoxy, C₅    alkoxy, C₅ alkoxy, C₆ alkoxy, C₇ alkoxy, C₈ alkoxy, C₉ alkoxy 1, C₁₀    alkoxy, or any combination thereof; C₆H₅OR₄; benzoyl isoleucine;    leucine aldehyde; phenyl optionally substituted with C₁-C₁₀ alkyl or    any of the individual alkyl groups of formula C₁-C₁₀ alkyl, C₁-C₁₀    alkoxy or any of the individual alkoxy groups of formula C₁-C₁₀    alkoxy, carbonyl, or amide; or benzyl optionally substituted with    C₁-C₁₀ alkyl or any of the individual alkyl groups of formula C₁-C₁₀    alkyl, C₁-C₁₀ alkoxy or any of the individual alkoxy groups of    formula C₁-C₁₀ alkoxy, carbonyl, or amide;-   R₄ is C₁-C₁₀ alkyl or any of the individual alkyl groups of formula    C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy or any of the individual alkoxy groups    of formula C₁-C₁₀ alkoxy, carbonyl, or amide; and-   B is at least one bisphosphonate optionally substituted with OH,    halogen, CH₃, NH₂, N-alkyl, or N-dialkyl; or a pharmaceutically    acceptable salt thereof, or a metabolite thereof.

A third embodiment includes the compound according to any one of thefirst and the second embodiments, wherein A is a gamma-secretaseinhibitor comprising the formula:

A fourth embodiment includes the compound according to any one of thefirst and the second embodiments, wherein the compound comprises one ormore stereoisomers of the formula:

-   wherein ‘R is H, CH₃, alkyl, halogen, CF₃, CN, OH, OCH₃, or O-alkyl;-   n is 1, 2, 3, 4, 5, 6, 7, 8, or 9;-   m is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; and-   X is H, OH, halogen, CH₃, NH₂, N-alkyl, or N-dialkyl; or a    pharmaceutically acceptable salt thereof, or a metabolite thereof.

In some embodiments, the one or more stereoisomers according to thefourth embodiments, comprise any one or more of the formula:

or a pharmaceutically acceptable salt thereof, or a metabolite thereof.Consistent with these embodiments, the one or more stereoisomers caninclude, but are not limited to, hydrogen(5S,8S,E)-5-((S)-sec-butyl)-8-isobutyl-11-methyl-3,6,15-trioxo-1-phenyl-17-phosphono-2-oxa-4,7,10,11,16-pentaazaheptadec-9-en-11-ium-17-yl)phosphonate.

A fifth embodiment includes the compound according to the fourthembodiment, wherein n is 1, 2, or 3, m is 0, and X is H.

A sixth embodiment includes the compound according to any one of thefirst to the fifth embodiments, or a pharmaceutically acceptable saltthereof, or a metabolite thereof, wherein said compound is a compound offormula:

A seventh embodiment includes the compound according to according to anyone of the first to the fifth embodiments, wherein said compound ishydrogen(5S,8S,E)-5-((S)-sec-butyl)-8-isobutyl-11-methyl-3,6,15-trioxo-1-phenyl-17-phosphono-2-oxa-4,7,10,11,16-pentaazaheptadec-9-en-11-ium-17-yl)phosphonate.

An eighth embodiment includes a method of reducing the growth of myelomacells and/or osteoclast differentiation, comprising the steps of:administering to a subject at least one therapeutically effective doseof a compound according to any one of the first to the seventhembodiments, or a pharmaceutically acceptable salt or metabolitethereof.

A nineth embodiment includes the method according to the eighthembodiment, further comprising the step of: administering to the subjectat least one therapeutically effective dose of parathyroid hormone.

A tenth embodiment includes the method according to any one of theeighth and the nineth embodiments, further comprising the step of:administering to the subject at least one therapeutically effective doseof at least one proteasome inhibitor.

An eleventh embodiment includes the method according to the tenthembodiment, wherein the at least one proteasome inhibitor compriseslactacystin, disulfiram, epigallocatechin-3-gallate, marizomib(salinosporamide A), oprozomib (ONX-0912), delanzomib (CEP-18770),epoxomicin, beta-hydroxy beta-methylbutyrate, bortezomib, carfilzomib,and/or ixazomib.

A twelfth embodiment includes the method according to any one of theeighth to the eleventh embodiments, wherein the subject comprises ahuman, an animal, a cell, and/or a tissue.

A thirteenth embodiment includes a method of treating a bone relateddisease, comprising the steps of: administering to a subject at leastone therapeutically effective dose of a compound according to any one ofthe first to the seventh embodiments, or a pharmaceutically acceptablesalt or metabolite thereof.

A fourteenth embodiment includes the method according to the thirteenthembodiment, further comprising the step of: administering to the subjectat least one therapeutically effective dose of parathyroid hormone.

A fifteenth embodiment includes the method according to any one of thethirteenth and the fourteenth embodiments, further comprising the stepof: administering to the subject at least one therapeutically effectivedose of at least one proteasome inhibitor.

A sixteenth embodiment includes the method according to the fifteenthembodiment, wherein the at least one proteasome inhibitor compriseslactacystin, disulfiram, epigallocatechin-3-gallate, marizomib(salinosporamide A), oprozomib (ONX-0912), delanzomib (CEP-18770),epoxomicin, beta-hydroxy beta-methylbutyrate, bortezomib, carfilzomib,and/or ixazomib.

A seventeenth embodiment includes the method according to any one of thethirteenth to the sixteenth embodiments, wherein the bone relateddisease comprises osteopenia, osteoporosis, rheumatoid arthritis,hematologic, gastrointestinal and pulmonary disease, autoimmunity,transplant rejection, multiple myeloma, bone cancer, brain cancer,breast cancer, endocrine cancer, gastrointestinal cancer, gynecologiccancer, prostate cancer, head and neck cancer, hematologic cancer, lungcancer, renal cell carcinoma, skin cancer, urologic cancer, rare cancer,skeletal or bone diseases, defects, and/or conditions associated with orinduced by glucocorticoid therapy.

An eighteenth embodiment includes the method according to any one ofclaims the thirteenth to the seventeenth embodiments, wherein thesubject comprises a human, an animal, a cell, and/or a tissue.

A nineteenth embodiment includes a method of treating a bone relateddisease, comprising the steps of: administering to a subject at leastone therapeutically effective dose of at least one agent that reducesand/or inhibits the activitiy of gamma-secretase, or a pharmaceuticallyacceptable salt or metabolite thereof; and at least one bisphosphonate,or a pharmaceutically acceptable salt or metabolite thereof.

A twentieth embodiment includes the method according to the nineteenthembodiment, further comprising the step of: administering to the subjectat least one therapeutically effective dose of parathyroid hormone.

A twenty first embodiment includes the method according to any one ofthe nineteenth and the twentieth embodiments, further comprising thestep of: administering to the subject at least one therapeuticallyeffective dose of at least one proteasome inhibitor.

A twenty second embodiment includes the method according to the twentyfirst embodiment, wherein the at least one proteasome inhibitorcomprises lactacystin, disulfiram, epigallocatechin-3-gallate, marizomib(salinosporamide A), oprozomib (ONX-0912), delanzomib (CEP-18770),epoxomicin, beta-hydroxy beta-methylbutyrate, bortezomib, carfilzomib,and/or ixazomib.

A twenty third embodiment includes the method according to any one ofthe nineteenth to the twenty second embodiments, wherein the at leastone agent that reduces and/or inhibits the activitiy of gamma-secretasecomprises a compound having the formula:

or a pharmaceutically acceptable salt thereof, or a metabolite thereof.

A twenty fourth embodiment includes the method according to any one ofthe nineteenth to the twenty third embodiments, wherein the bone relateddisease comprises osteopenia, osteoporosis, rheumatoid arthritis,hematologic, gastrointestinal and pulmonary disease, autoimmunity,transplant rejection, multiple myeloma, bone cancer, brain cancer,breast cancer, endocrine cancer, gastrointestinal cancer, gynecologiccancer, prostate cancer, head and neck cancer, hematologic cancer, lungcancer, renal cell carcinoma, skin cancer, urologic cancer, rare cancer,skeletal or bone diseases, defects, and/or conditions associated with orinduced by glucocorticoid therapy.

A twenty fifth embodiment includes the method according to any one ofthe nineteenth to the twenty fourth embodiments, wherein the subjectcomprises a human, an animal, a cell, and/or a tissue.

A twenty sixth embodiment includes the method accoding to any one of theeighth to the twenty fifth embodiments, wherein the therapeuticallyeffective dose of parathyroid hormone, is on the order of between about0.01 µg to about 1000 µg and the dose of the compound is administered tothe patient at least once per day. Consistent with these embodiments,the therapeutically effective dose of parathyroid hormone includes, butis not limited to, on the order of between: about 0.01 µg to about 1000µg; about 0.01 µg to about 500 µg; about 0.01 µg to about 200 µg; about0.01 µg to about 150 µg; about 0.01 µg to about 100 mg; about 0.01 µg toabout 80 µg; about 0.01 µg to about 50 µg; about 0.05 µg to about 100mg; about 0.05 µg to about 80 µg; about 0.05 µg to about 50 µg; about0.1 µg to about 100 µg; about 0.1 µg to about 50 µg; about 0.2 µg toabout 100 µg; about 0.2 µg to about 50 µg; about 0.5 µg to about 100 µg;about 0.5 µg to about 50 µg; about 10 µg to about 200 µg; about 50 µg toabout 200 µg; about 10 µg to about 100 µg; about 50 µg to about 100 µg;about 100 µg to about 150 µg; about 10 µg, about 20 µg, about 30 µg,about 40 µg, about 50 µg, about 60 µg, about 70 µg, about 80 µg, about90 µg, about 100 µg, and/or any combination thereof.

A twenty seventh embodiment includes the method accoding to any one ofthe eighth to the twenty sixth embodiments, wherein the therapeuticallyeffective dose of a compound according to any one of the first to theseventh embodiments, or a pharmaceutically acceptable salt or metabolitethereof, is on the order of between about 0.001 µg to about 1000 µg andthe dose of the compound is administered to the patient at least onceper day. Consistent with these embodiments, the therapeuticallyeffective dose of a compound according to any one of the first to theseventh embodiments, or a pharmaceutically acceptable salt or metabolitethereof, includes, but is not limited to, on the order of between: about0.001 µg to about 1000 µg; about 0.001 µg to about 500 µg; about 0.001µg to about 200 µg; about 0.001 µg to about 150 µg; about 0.001 µg toabout 100 mg; about 0.001 µg to about 80 µg; about 0.001 µg to about 60µg; about 0.005 µg to about 100 mg; about 0.005 µg to about 80 µg; about0.005 µg to about 50 µg; about 0.01 µg to about 100 µg; about 0.01 µg toabout 50 µg; about 0.02 µg to about 100 µg; about 0.02 µg to about 50µg; about 0.05 µg to about 100 µg; about 0.05 µg to about 50 µg; about 1µg to about 200 µg; about 1 µg to about 150 µg; about 1 µg to about 100µg; about 1 µg to about 50 µg; about 0.01 µg, about 0.1 µg, about 1 µg,about 5 µg, about 10 µg, about 20 µg, about 30 µg, about 40 µg, about 50µg, about 60 µg, about 70 µg, about 80 µg, about 90 µg, about 100 µg,and/or any combination thereof.

A twenty eighth embodiment includes the method accoding to any one ofthe eighth to the twenty sixth embodiments, wherein the compoundaccording to any one of the first to the seventh embodiments, or apharmaceutically acceptable salt or metabolite thereof is formulated foradministration to the subject for delivery orally, subcutaneously,intramuscularly, intradermally, intranasally, topically, transdermally,parenterally, gastrointestinally, transbronchially, transalveolarly,and/or mucosally.

A twenty nineth embodiment includes the method accoding to any one ofthe eighth to the twenty eighth embodiments, wherein parathyroid hormoneand/or the at least one proteasome inhibitor is formulated foradministration to the subject for delivery orally, subcutaneously,intramuscularly, intradermally, intranasally, topically, transdermally,parenterally, gastrointestinally, transbronchially, transalveolarly,and/or mucosally.

A thirtieth embodiment includes a composition comprising the compoundaccording to any one of the first to the seventh embodiments, or apharmaceutically acceptable salt or metabolite thereof and at least oneagent that induces bone anabolism.

A thirty first embodiment includes the composition according to thethirtieth embodiment, wherein the at least one agent that induces boneanabolism includes, but is not limited to, parathyroid hormone.

A thirty second embodiment includes the composition according to thethirtieth and the thirty first embodiments, wherein the compositionincreases bone mass in a subject.

A thirty third embodiment includes the composition according to any oneof the thirtieth and the thirty second embodiments, wherein the compoundaccording to any one of the first to the seventh embodiments andparathyroid hormone are present in the composition in a concentrationratio such that the composition exhibits synergy. Consistent with theseembodiments, the concentration ratio of the compound according to anyone of the first to the seventh embodiments and parathyroid hormone canbe from about 100:1 to about 1:100, from about 90:1 to about 1:90, fromabout 80:1 to 1:80, from about 50:1 to about 1:50, from about 20:1 toabout 1:20, from about 10:1 to about 1:10, from about 8:1 to 1:8, fromabout 5:1 to about 1:5, from about 2:1 to about 1:2, from about 1:1 toabout 1:1, about 100:1, about 50:1, about 20:1, about 10:1, about 5:1,about 2:1, about 1:1, about 1:2, about 1:5, about 1:10, about 1:20,about 1:50, about 1:100, or any combination thereof.

A thirty fourth embodiment includes the composition according to any oneof the thirtieth to the thirty third embodiments, further comprising atleast one proteasome inhibitor.

A thirty fifth embodiment includes the composition according to thethirty fourth embodiments, wherein the at least one proteasome inhibitorcomprises lactacystin, disulfiram, epigallocatechin-3-gallate, marizomib(salinosporamide A), oprozomib (ONX-0912), delanzomib (CEP-18770),epoxomicin, beta-hydroxy beta-methylbutyrate, bortezomib, carfilzomib,and/or ixazomib.

A thirty sixth embodiment includes the composition according to any oneof the thirtieth to the thirty fifth embodiments, wherein the subjectcomprises a human, an animal, a cell, and/or a tissue.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A. Graphs illustrating the effect on Notch target gene expression.In vitro, the control unconjugated GSI decreased Notch target geneexpression (Hes1-Hey1) but BT-GSI-XII had no effect.

FIG. 1B. A graph illustrating the effect on Notch target geneexpression. GSI-XII and BT-GSI-XII preincubated at low pH to mimic theacidic conditions in resorption sites exhibit equal inhibition of Notchtarget gene expression.

FIG. 1C. A schematic drawing illustring an exemplary ex vivo system. Exvivo, both GSI-XII and BT-GSI-XII (non-preincubated) similarly decreasedHes⅕ expression in whole bone organ cultures that reproduce conditionsin the bone microenvironment.

FIG. 1D. Graphs illustrating the effect of BT-GSI-XII on Bone MineralDensity (BMD). BT-GSI-XII treated mice exhibited higher total (3%),femoral (4%), and spinal (7%) BMD compared to control mice.

FIG. 1E. Graphs illustrating the effect of BT-GSI-XII on the boneformation markers. BT-GSI-XII did not affect the circulating levels ofthe bone formation marker P1NP, but decreased serum CTX by 40%, a markerof bone resorption.

FIG. 2A. Graphs illustrating the effect on Notch target genes. Micetreated with BT-GSI exhibited decreased Hes5, Hes7, Hey2, and HeyLexpression in bone, but not in brain or gut, compared to vehicle-treatedmice.

FIG. 2B. Graphs illustrating the effect on Notch target genes. Micetreated with BT-GSI exhibited decreased Hes5, Hes7, Hey2, and HeyLexpression in bone, but not in brain or gut, compared to vehicle-treatedmice. Further, BT-GSI-XII did not increase the expression in the gut ofApsidin, a biomarker of gastrointestinal toxicity.

FIG. 3A. Graphs illustrating the effect on pro- andanti-osteoclastogenic cytokines. Mice treated with BT-GSI-XII exhibiteincreased Opg mRNA expression in bone, thus decreasing the Rankl/Opgratio.

FIG. 3B. Graphs illustrating the effect on osteoblast markers. Theexpression of osteoblast markers remained unchanged by BT-GSI-XII.

FIG. 4 . Exemplary structural formula of some of UR-15 (also referred toas BT-GSI-XII) analogs.

FIG. 5 . Exemplary structural formula of some of UR-15 analogs.

FIG. 6 . Exemplary structural formula of some of UR-15 analogs.

FIG. 7 . Exemplary structural formula of some of UR-15 analogs.

FIG. 8 . Exemplary structural formula of some of UR-15 analogs.

FIG. 9 . Exemplary structural formula of some of stereoisomers.

FIG. 10A. Graphs illustrating the effect of daily injections of PTH(iPTH) and BT-GSI-XII on Notch target gene expression in bone.

FIG. 10B. Graphs illustrating the effect of daily injections of PTH(iPTH) and BT-GSI-XII on Notch target gene expression in brain.

FIG. 10C. Graphs illustrating the effect of daily injections of PTH(iPTH) and BT-GSI-XII on Notch target gene expression in gut.

FIG. 11A. Graphs illustrating the effect of daily injections of PTH(iPTH) and BT-GSI-XII on Bone Mineral Density (BMD).

FIG. 11B. Pictures illustrating the effect of daily injections of PTH(iPTH) and BT-GSI-XII on bone mass.

FIG. 11C. Graphs illustrating the effect of daily injections of PTH(iPTH) and BT-GSI-XII on cortical area fraction (Ct.A/Tt.A) and corticalthickness (Ct.Th.).

FIG. 11D. Graphs illustrating the effect of daily injections of PTH(iPTH) and BT-GSI-XII on bone volume fraction (BV/TV), trabecularthickness (Tb.Th.), trabecular number (Tb.N.), and trabecular spacing(Tb.Sp) in cancellous femur and/or in cancellous vertebrae.

FIG. 12A. A graph illustrating the effect of daily injections of PTH(iPTH) and BT-GSI-XII on the serum levels of the bone resorption markerC-terminal telopeptide (CTX).

FIG. 12B. (Left) Graphs illustrating the effect of daily injections ofPTH (iPTH) and BT-GSI-XII on the level of osteoclast number per bonesurface (Oc.N/BS) and osteoclast surface per bone surface (Oc.S/BS) incancellous bone. (Right) Representative TRAP staining illustrating theeffect of daily injections of PTH (iPTH) and BT-GSI-XII on osteocalstsin cancellous bone.

FIG. 12C. Graphs illustrating the effect of daily injections of PTH(iPTH) and BT-GSI-XII on the mRNA gene expression of pro- andanti-osteoclastogenic cytokines.

FIG. 12D. A graph illustrating the effect of BT-GSI-XII on the number ofosteclast.

FIG. 13A. A graph illustrating the effect of daily injections of PTH(iPTH) and BT-GSI-XII on the serum levels of the bone formation markerprocollagen type 1 amino-terminal propeptide (P1NP).

FIG. 13B. (Left) Graphs illustrating the effect of daily injections ofPTH (iPTH) and BT-GSI-XII on the level of mineralizing surface per bonesurface (MS/BS), mineral apposition rate (MAR), bone formation rate perunit of bone surface (BFR/BS). (Right) Representative alizarin Red /calcein labels staining illustrating the effect of daily injections ofPTH (iPTH) and BT-GSI-XII on cancellous vertebrae.

FIG. 13C. Graphs illustrating the effect of daily injections of PTH(iPTH) and BT-GSI-XII on the level of osteoblast number per bone surface(Ob.N/BS) and osteoblast surface per bone surface (Ob.S/BS) incancellous bone.

FIG. 13D. Graphs illustrating the effect of daily injections of PTH(iPTH) and BT-GSI-XII on the level of Wnt target gene expression.

FIG. 13E. Graphs illustrating the effect of daily injections of PTH(iPTH) and BT-GSI-XII on the level of osteoblast markers.

DESCRIPTION

For the purposes of promoting an understanding of the principles of thenovel technology, reference will now be made to the preferredembodiments thereof, and specific language will be used to describe thesame. It will nevertheless be understood that no limitation of the scopeof the novel technology is thereby intended, such alterations,modifications, and further applications of the principles of the noveltechnology being contemplated as would normally occur to one skilled inthe art to which the novel technology relates are within the scope ofthis disclosure and the claims.

As used herein, unless explicitly stated otherwise or clearly impliedotherwise the term ‘about’ refers to a range of values plus or minus 10percent, e.g. about 1.0 encompasses values from 0.9 to 1.1.

The term, “treating” as used herein unless stated or implied otherwise,includes administering to a human or an animal patient at least one doseof a compound, treating includes preventing or lessening the likelihoodand/or severity of at least one disease as well as limiting the lengthof an illness or the severity of an illness, treating may or may notresult in a cure of the disease.

As used herein, unless explicitly stated otherwise or clearly impliedotherwise the terms ‘therapeutically effective dose,’ ‘therapeuticallyeffective amounts,’ and the like, refer to a portion of a compound thathas a net positive effect on health and well being of a human or otheranimal. Therapeutic effects may include an improvement in longevity,quality of life and the like these effects also may also include areduced susceptibility to developing disease or deteriorating health orwell being. The effects may be immediate realized after a single doseand/or treatment or they may be cumulative realized after a series ofdoses and/or treatments. A “therapeutically effective amount” in generalmeans the amount that, when administered to a subject or animal fortreating a disease, is sufficient to affect the desired degree oftreatment for the disease.

As used herein, “inhibition” or “inhibitory activity” each encompasswhole or partial reduction of activity or effect of an enzyme or alland/or part of a pathway that includes an enzyme that is effected eitherdirectly or indirectly by the inhibitor or a pathway that is effectedeither directly or indirectly by the activity of the enzyme which iseffected either directly or indirectly by the inhibitor.

As used herein, the term “pharmaceutically acceptable salt” is definedas a salt wherein the desired biological activity of the inhibitor ismaintained and which exhibits a minimum of undesired toxicologicaleffects. Non-limiting examples of such a salt are (a) acid additionsalts formed with inorganic acids (e.g., hydrochloric acid, hydrobromicacid, sulphuric acid, phosphoric acid, nitric acid, and the like), andsalts formed with organic acids (such as e.g. acetic acid, oxalic acid,tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid,tannic acid, palmitic acid, polyglutamic acid, naphthalene sulphonicacid, naphthalene disulphonic acid, polygalacturonic acid and the like);(b) base additional salts formed with metal cations such as zinc,calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel,cadmium, sodium, potassium and the like, or with a cation formed fromammonia, N,N-dibenzylethylenediamine, D-glucosamine, tetraethylammoniumor ethylenediamine; or (c) combinations of (a) and (b); e.g. a zinctannate or the like.

Pharmaceutically acceptable salts include salts of compounds of theinvention that are safe and effective for use in mammals and thatpossess a desired therapeutic activity. Pharmaceutically acceptablesalts include salts of acidic or basic groups present in compounds ofthe invention. Pharmaceutically acceptable acid addition salts include,but are not limited to, hydrochloride, hydrobromide, hydroiodide,nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate,acetate, lactate, salicylate, citrate, tartrate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucaronate, saccharate, formate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzensulfonate, p-toluenesulfonateand pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts.Certain compounds of the invention may form pharmaceutically acceptablesalts with various amino acids. Suitable base salts include, but are notlimited to, aluminum, calcium, lithium, magnesium, potassium, sodium,zinc, and diethanolamine salts. For additional information on somepharmaceutically acceptable salts that can be used to practice theinvention please reviews such as Berge, et al., 66 J. PHARM. SCI. 1-19(1977), Haynes, et al, J. Pharma. Sci., Vol. 94, No. 10, October 2005,pgs. 2111-2120 and See, e.g., P. Stahl, et al., HANDBOOK OFPHARMACEUTICAL SALTS: PROPERTIES, SELECTION AND USE, (VCHA/Wiley-VCH,2002); S.M. Berge, et al., “Pharmaceutical Salts,” Journal ofPharmaceutical Sciences, Vol. 66, No. 1, January 1977.

Pharmaceutical formulation: The compounds of the invention and theirsalts may be formulated as pharmaceutical compositions foradministration. Such pharmaceutical compositions and processes formaking the same are known in the art for both humans and non-humanmammals. See, e.g., REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, (A.Gennaro, et al., eds., 19^(th) ed., Mack Publishing Co., 1995).Formulations can be administered through various means, including oraladministration, parenteral administration such as injection(intramuscular, subcutaneous, intravenous, intraperitoneal) or the like;transdermal administration such as dipping, spray, bathing, washing,pouring-on and spotting-on, and dusting, or the like. Additional activeingredients may be included in the formulation containing a compound ofthe invention or a salt thereof.

The pharmaceutical formulations of the present invention include thosesuitable for oral, parenteral (including subcutaneous, intradermal,intramuscular and intravenous) and rectal administration. Theformulations may be presented in unit dosage form and may be prepared byany of the methods well known in the art of pharmacy. All methodsinclude the step of bringing into association the active ingredient,i.e., the compound or salt of the present invention, with the carrier.In general, the formulations are prepared by uniformly and intimatelybringing into association the active ingredient with a liquid carrieror, a finely divided solid carrier or both, and then, if necessary,forming the associated mixture into the desired formulation.

The pharmaceutical formulations of the present invention suitable fororal administration may be presented as discrete units, such as acapsule, cachet, tablet, or lozenge, each containing a predeterminedamount of the active ingredient; as a powder or granules; as a solutionor a suspension in an aqueous liquid or non-aqueous liquid such as asyrup, elixir or a draught, or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The formulation may also be a bolus,electuary or paste.

The pharmaceutical formulations of the present invention suitable forparenteral administration include aqueous and non-aqueous sterileinjection solutions, and may also include an antioxidant, buffer, abacteriostat and a solution which renders the composition isotonic withthe blood of the recipient, and aqueous and non-aqueous sterilesuspensions which may contain, for example, a suspending agent and athickening agent. The formulations may be presented in a singleunit-dose or multi-dose containers, and may be stored in a lyophilizedcondition requiring the addition of a sterile liquid carrier prior touse.

Pharmaceutically acceptable carrier: Pharmaceutically acceptablecarrier, unless stated or implied otherwise, is used herein to describeany ingredient other than the active component(s) that maybe included ina formulation. The choice of carrier will to a large extent depend onfactors such as the particular mode of administration, the effect of thecarrier on solubility and stability, and the nature of the dosage form.

A tablet may be made by compressing or moulding the active ingredientwith the pharmaceutically acceptable carrier. Compressed tablets may beprepared by compressing in a suitable machine the active ingredient in afree-flowing form, such as a powder or granules, in admixture with, forexample, a binding agent, an inert diluent, a lubricating agent, adisintegrating and/or a surface active agent. Moulded tablets may beprepared by moulding in a suitable machine a mixture of the powderedactive ingredient moistened with an inert liquid diluent. The tabletsmay optionally be coated or scored and may be formulated so as toprovide slow or controlled release of the active ingredient.

The term, “synergism” or “synergy” refers to an interaction of two ormore factors such that the effect when combined is greater than thepredicted effect based on the response of each factor appliedseparately.

As used herein, “bone related diseases” include, but are not limited to,osteopenia, osteoporosis, rheumatoid arthritis, hematologic,gastrointestinal and pulmonary diseases, autoimmunity, transplantrejection, multiple myeloma, bone cancer, brain cancer, breast cancer,endocrine cancer, gastrointestinal cancer, gynecologic cancer, prostatecancer, head and neck cancer, hematologic cancer, lung cancer, renalcell carcinoma, skin cancer, urologic cancer, rare cancers, and/orskeletal or bone diseases, defects, and/or conditions associated with orinduced by glucocorticoid therapy.

As used herein, “proteasome inhibitors” include, but are not limited to,lactacystin, disulfiram, epigallocatechin-3-gallate, marizomib(salinosporamide A), oprozomib (ONX-0912), delanzomib (CEP-18770),epoxomicin, beta-hydroxy beta-methylbutyrate, bortezomib, carfilzomib,and ixazomib.

Multiple myeloma is a plasma cell malignancy characterized by expansionof monoclonal plasma cells in the bone marrow (BM) and the presence ofosteolytic lesions. Multiple myeloma has the highest incidence of boneinvolvement among malignant diseases. Multiple myeloma patients presentwith severe bone pain caused by osteolytic lesions that rarely heal. Theosteolytic lesions result from increased bone resorption and concomitantlong-term suppression of bone formation. The bone and BMmicroenvironment is a major contributor to tumor growth and bonedestructive process in multiple myeloma. See e.g., Jesus Delgado-Calleet. al., Role of osteocytes in multiple myeloma bone disease, CURR OPINSUPPORT PALLIAT CARE, 2014; 8(4): 407-413.

Notch signaling plays a critical role in cell-to-cell communicationamong bone and bone marrow cells under physiological conditions and itfavors growth and survival of cancer cells in bone. However, geneticmanipulation of this pathway rendered different bone phenotypesdepending on the Notch component (ligands, receptors, target genes), thecell lineage, or differentiation stage being targeted; and skeletalphenotypes result from combined developmental and postnatal effects. Invitro and in vivo studies demonstrated that systemic inhibition of Notchsignaling using gamma-secretase inhibitors (GSIs) decreases the growthof myeloma cells and osteoclast differentiation. However, the use ofGSIs in the clinic is limited by the presence of severe adverse sideeffects as fatigue, skin disorders, and acute gastrointestinal toxicity.

Bone fragility leading to fractures and disability is associated withthe bone disease caused by glucocorticoid excess, sex steroiddeficiency, and advanced age. Increased osteoblast apoptosis is at leastpartially responsible for the decreased bone formation rate associatedwith the osteopenia induced by glucocorticoid excess. Studies usingosteocytic MLO-Y4 cells and primary murine calvaria cells demonstratedthat bisphosphonates can inhibit apoptosis induced by the glucocorticoiddexamethasone, the inhibitor of DNA repair etoposide that blockstopoisomerase II activity, or TNFα, an activator of death receptors.Bisphosphonates can stop bone loss by inhibiting the activity or byincreasing the rate of apoptosis of bone resorbing osteoclasts. Seee.g., Teresita Bellido et. al., Novel actions of bisphosphonates inbone: Preservation of osteoblast and osteocyte viability, BONE, 2011;49(1): 50-55.

Sclerostin, the product of the Sost gene, is expressed and secretedprimarily by osteocytes and inhibits bone formation by osteoblasts,fueled research attempting to identify regulators of this gene as wellas other osteocyte products that impact the function of osteoblasts andosteoclasts. Parathyroid hormone (PTH), a central regulator of bonehomeostasis, can inhibit sclerostin expression. See e.g., TeresitaBellido et. al., Effects of PTH on osteocyte function, BONE, 2013;54(2): 250-257.

Synthesis Procedure

Tetraethyl (E)[4-(2-Benzylidene-1-Methylhydrazinyl)-Butanamidomethylene]Bisphosphonate

A 10 mL round bottom flask was charged with dry DCM (1 mL),bisphosphonate amine (100 mg, 0.33 mmol, 1 equiv), and 2,6-lutidine(70.6 mg, 0.66 mmol, 2 equiv) at 0° C. under and Ar atmosphere.4-bromobutyryl chloride (72 mg, 0.38 mmol, 1.2 equiv) was added into thereaction mixture. After stirring for 5 min, benzaldehydeN-methylhydrazone (133 mg, 0.99 mmol, 3 equiv) was added, and thereaction mixture was allowed to warm to rt and stir for 15 hr. The crudewas purified directly by column chromatography with silica gel usingethyl acetate to elute the forerunning impurities then usingacetone-ethyl acetate gradient (1:4 to 1:3) to afford the product (117mg, 70%) as pale yellow oil.

R_(f) = 0.43 (Ethyl Acetate-Acetone, 3:1). I₂ active. ¹H NMR (400 MHz,CDCl₃): δ 7.52 (d, J = 7.6 Hz, 1H), 7.32 - 7.11 (m, 3H), 6.37 (s, 1H),5.06 (td, J = 21.8, 10.0 Hz, 1H), 4.17 (d, J = 3.2 Hz, 8H), 3.65 - 3.26(m, 2H), 2.88 (s, 3H), 2.45 - 2.37 (dd, J = 29.1, 22.0 Hz, 2H), 2.14 -2.01 (dd, J = 30.3, 23.3 Hz, 2 H), 1.31 (t, J = 8.5 Hz, 12H). ¹³C NMR(101 MHz, CDCl₃): δ 171.81, 136.91, 131.66, 128.40, 127.11, 125.42,63.55, 63.42, 57.30, 43.34, 37.55, 33.32, 32.72, 23.52, 16.29. ³¹P NMR(162 MHz, CDCl₃) δ 16.49, 13.24, 13.08. IR (cm⁻¹): 3248.13, 2981.95,2908.65, 1674.21, 1529.55. LC-MS (APCI) m/z Calcd for C₂₁H₃₇N₃O₇P₂(M+H⁺): 505.21. Found: 505.87.

Benzyl (2S,3S)-1-[(S,E)-1-[2-(4-(Bis-Diethoxyphosphorylmethylene)Amino)-4-Oxobutyl)-2-Methylhydrazinylidene)-4-Methylpentan-2-Yl)Amino)-3-Methyl-1-Oxopentan-2-yl)Carbamate

A 10 mL round bottom flask was charged with tetraethyl (E)[4-(2-benzylidene-1-methylhydrazinyl)-butanamidomethylene]bisphosphonate (115 mg, 0.227 mmol, 1 equiv), pyridine (72 mg, 0.91mmol, 4 equiv) in ethanol (1.5 mL). Hydroxylamine hydrochloride (63.2mg, 0.91 mmol, 4 equiv) was added and the reaction mixture was thenwarmed to 70° C. for 14 hr. After cooling to rt, the reaction mixturewas quenched with saturated NaHCO₃ (0.5 mL), and then basified with 1 MNaOH solution. The reaction mixture was extracted with ethyl acetate (15mL) for twice, and the aqueous layer was concentrated under vacuum toobtain white solid. Washed the solid with DCM then concentrated toobtain colorless oil (82 mg). Without further purification, thecolorless oil was added into the solution of GSI XII (82 mg, 0.227 mmol,1 equiv) in DCM (1.5 mL) with acetic acid (2.9 mg, 0.045 mmol, 0.2equiv). The reaction mixture was allowed to reflux at 45° C. over-nightfor 12 hr. After cooling to rt, the crude was purified directly bycolumn chromatography with silica gel using ethyl acetate to remove theeasily eluted impurities then using methanol-ethyl acetate gradient(1:9) to afford the product (37 mg, 28%) as pale yellow oil.

R_(f) = 0.32 (Acetate-Methanol, 9:1). CAM active. ¹H NMR (400 MHz,CDCl₃): δ 7.68 (d, J = 12.0 Hz, 1H), 7.49 (d, J = 24.7 Hz, 1H), 7.33 (m,5H), 6.83 (s, 1H), 6.59 (s, 1H), 5.11 (s, 2H), 4.56 (s, 1H), 4.20 (s,8H), 4.10 - 3.95 (m, 1H), 3.15 (s, 1H), 2.68 (s, 3H), 2.29 (s, 2H), 1.89(d, J = 6.1 Hz, 3H), 1.67 (d, J = 7.1 Hz, 2H), 1.49 (s, 4H), 1.47 - 1.25(m, 12H), 1.09 - 0.78 (m, 12H). ¹³C NMR (101 MHz, CDCl₃): δ 172.97,172.32, 171.06, 170.94, 170.61, 156.91, 156.36, 136.47, 134.01, 133.62,128.38, 127.94, 127.81, 127.29, 66.55, 64.39, 63.36, 61.98, 59.80,58.07, 56.67, 56.55, 49.74, (t, J_(P-C) = 59.1 Hz), 44.65, 44.44, 43.18,42.90, 42.73, 41.72, 38.67, 37.43, 37.26, 36.57, 34.11, 33.21, 24.77,24.72, 24.60, 23.27, 23.02, 22.83, 22.67, 22.56, 22.23, 16.25, 15.49,11.48, 11.27. ³¹P NMR (162 MHz, CDCl₃) δ 13.21. IR (cm⁻¹): 3271.33,2960.78, 1704.14, 1656.88, 1530.54. LC-MS (APCI) m/z Calcd forC₃₄H₆₂N₅O₁₀P₂ (M+H⁺): 762.39. Found: 762.20.

Hydrogen(5S,8S,E)-5-((S)-Sec-Butyl)-8-Isobutyl-11-Methyl-3,6,15-Trioxo-1-Phenyl-17-Phosphono-2-Oxa-4,7,10,11,16-Pentaazaheptadec-9-En-11-Ium-17-Yl)Phosphonate

A dry 10 mL round bottom flask was charged with a solution of theforegoing hydrazone (1 eq, 0.046 mmol, 32 mg) in dry DCM (0.4 mL) underAr at 0° C. Trimethylsilyl bromide (TMSBr) (6 eq, 0.28 mmol, 42.2 mg)was added dropwise with magnetic stirring. After addition was complete,the reaction mixture was allowed to stir and warm to rt overnight. Thereaction mixture was then concentrated in vacuum and kept under highvacuum for 10 min to afford a brown solid. And methanol (3 mL) was addedto dissolve the solid, and the resulting solution was concentrated invacuum. This procedure was repeated 3 additional times. The solid wasdried under high vacuum affording 28.7 mg of desired product as a paleyellow solid (99%).

¹HNMR (500 MHz, MeOD): δ 7.66 - 7.56 (m, 1H), 7.52 (s, 1H), 7.31 (m,4H), 5.45 (s, 2H), 5.05 (s, 1H), 4.52 (s, 1H), 4.11 (s, 1H), 3.66 - 3.52(m, 1H), 3.18 (d, J = 6.9 Hz, 1H), 3.02 (s, 2H), 2.66 (s, 3H), 2.45 (s,2H), 2.30 (s, 1H), 1.95 (s, 2H), 1.27 (t, J = 11.4 Hz, 4H), 1.14 - 0.67(m, 12H). ¹³C NMR (101 MHz, CDCl₃): δ 172.97, 172.32, 171.06, 170.94,170.61, 156.91, 156.36, 136.47, 134.01, 133.62, 128.38, 127.94, 127.81,127.29, 66.55, 64.39, 63.36, 61.98, 59.80, 58.07, 56.67, 56.55, 49.19(t, J = 59.1 Hz), 44.65, 44.44, 43.18, 42.90, 42.73, 41.72, 38.67,37.43, 37.26, 36.57, 34.11, 33.21, 24.77, 24.72, 24.60, 23.27, 23.02,22.83, 22.67, 22.56, 22.23, 16.25, 15.49, 11.48, 11.27. ³¹P NMR (162MHz, CDCl₃) δ 15.44. IR (cm⁻¹): 3201.83, 3026.31, 2960.73, 2931.80,2875.86, 2360.87, 1668.43, 1537.27. Thermo-MS (ESI) m/z: Calcd forC₂₇H₅₃N₅O₁₁P₂ (M+MeOH+4H⁺): 685.32. Found: 685.40.

EXAMPLES

To at least reduce or circumbent the side effects associated with paninhibition of Notch signaling using GSIs, novel Notch inhibitors weresynthesized by linking, for example, GSI-XII to an inactivebone-targeting molecule (BT). The BT portion of the conjugate is thoughto direct the conjugate to bone where the linker is cleaved nearosteoclasts, thus releasing GSI. Forms of BT useful for the applicationcan include, but are not limited to, bisphosphonates optionallysubstituted with OH, halogen, CH₃, NH₂, N-alkyl, or N-dialkyl. See, forexample, FIGS. 4-8 . BT-GSI was designed to direct the conjugate to bonewhere the linker is cleaved by, for example, acid produced byosteoclasts, thereby releasing GSI.

As used herein, unless explicitly stated otherwise or clearly impliedotherwise the compound “BT-GSI-XII” comprises the formula:

Referring now to FIG. 1A, the control unconjugated GSI decreased Notchtarget gene expression (Hes1) in 5TGM1 myeloma cells, but BT-GSI-XII hadno effect in vitro. However, when both GSI-XII and BT-GSI-XII werepre-incubated at low pH to mimic the acidic conditions in resorptionsites, equal inhibition of Notch target gene expression was observed inmyeloma cells (FIG. 1B). Referring now to FIG. 1C, ex vivo, both GSI-XIIand BT-GSI-XII (non-pre-incubated) similarly decreased Hes⅕ expressionin whole bone organ cultures that reproduce conditions present in thebone microenvironment. In vivo, administration of BT-GSI-XII (5 µg/g,3xwk) to normal 4-month old female mice for 2 weeks was well toleratedand no skin issues were observed. Referring now to FIG. 1D, BT-GSI-XIItreated mice exhibited higher total (3%), femoral (4%), and spinal (7%)BMD compared to control mice. BT-GSI-XII did not affect the circulatinglevels of the bone formation marker P1NP, but decreased serum CTX by40%, a marker of bone resorption. (FIG. 1E). Referrning now to FIG. 2 ,mice treated with BT-GSI exhibited decreased Hey2, Hes5, and Hes7 mRNAexpression in whole bone preparations, but not in brain or gut, comparedto vehicle-treated mice. Further, BT-GSI did not increase Apsidinexpression in the gut, a biomarker of gastrointestinal toxicity.BT-GSI-treated mice had decreased serum levels of CTX (-40%), a boneresorption marker, and upregulated Opg mRNA expression in bone, therebydecreasing the Rankl/Opg ratio (FIG. 3 ). Consistent with thesefindings, BT-GSI-XII treated mice exhibited a 50% decreased in the bonesurface covered by osteoclasts compared to control mice. BT-GSI-XIItreated mice exhibited higher total (+3%), femoral (+4%), and spinal(+7%) BMD compared to control mice. Further, microCT analysis revealedthat mice receiving BT-GSI-XII had increased cancellous bone volume(+25%) and trabecular thickness (+10%) compared to vehicle-treated mice.Serum levels of the bone formation marker P1NP, bone formation andmineral apposition rates, number of osteoblasts, and the expression ofosteoblast markers, including Wnt target genes and the osteocyte-derived Sost/Sclerostin remained unchanged by BT-GSI-XII.

Taken together, these findings appear to demonstrate that BT-GSIs (e.g.,BT-GSI-XII) induce bone specific Notch inhibition, reduce osteoclastformation without affecting osteoblast activity, and lacks gut toxicity.Further, a BT-GSI can circumvent the deleterious side effects that limitthe use of this class of inhibitors. Further, BT-GSIs inhibit boneresorption and favors bone gain. Thus, BT-GSI can be a promisingapproach to inhibit the growth of myeloma cells and improve skeletaldisease in myeloma patients by inhibiting resorption.

Daily injections of PTH (iPTH) are thought to cause bone anabolism byincreasing osteoblast number and function. However, iPTH can alsoincrease bone resorption, which can limit bone gain. iPTH activatesNotch signaling in osteocytes; and bone-targeted Notch inhibition usinga γ-secretase inhibitor (GSI) conjugated to an alendronate-modifiedbone-targeting molecule (BT-GSI) decreases Notch signaling in bone, andreduces CTX (-40%) while preserving bone formation, leading to increasesin BMD (4-7%) and cancellous bone volume (BV/TV; 30%).

To investigate whether a combination of iPTH (anabolic) and BT-GSI(anti-catabolic) increases bone mass to a higher extent than eitheragent alone, BT-GSI (5 mg/kg, 3x/wk) or saline was co-administered withiPTH (100 ng/g/day) or vehicle for 2 wks to 4-mo-old mice (n=10/group).Referring now to FIG. 10 , iPTH increased expression of the Notch targetgenes Hes1/5/7 and Hey1/L in bone, and co-administration of BT-GSIdecreased the iPTH-induced elevated expression of Hes1/5/7 and Hey1/L toa level comparable to the control mice receiving vehicle injections(FIG. 10A). The expression of Notch target genes in brain or gutremained unchanged by either iPTH, BT-GSI, or the combination (FIGS. 10Band 10C) iPTH increased total (7%) and femoral (13%), and preservedspinal BMD (0%); and co-administration of BT-GSI potentiated theincrease at all bone sites (10, 17, and 7%, respectively) (FIG. 11 ).Co-administration of BT-GSI exhibited additional 25% increase incancellous BV/TV (L4 and distal femur) and trabecular thickness whencompared to the iPTH-induced increase. However, the co-administration ofBT-GSI did not alter the iPTH-induced increase in cortical bone area(8%).

Referring to FIG. 12 , co-administration of BT-GSI decreasediPTH-induced elevated serum CTX (30%) and osteoclast number/surface(25%) levels to the values that are less than those exhibited in controlmice receiving vehicle (FIGS. 12A-B). The iPTH-induced elevatedRankl/Opg expression ratio in bone (1.5-fold) remained unchanged by theco-administration of BT-GSI (FIG. 12C). No changes were found in M-Csfexpression. Additionally, unconjugated GSI reduced Rankl-inducedosteoclast differentiation in vitro with an EC₅₀ ~0.1 µM (FIG. 12D).Further, the bone-targeting molecule alone required a dose 10 timeshigher than GSI to decreased osteoclastogenesis. Referring now to FIG.13 , co-administration of BT-GSI decreased iPTH-induced elevated serumP1NP (e.g., about 30% decrease); however, the co-administration ofBT-GSI maintained the iPTH-induced elevated bone formation rate (20%)and osteoblast number/surface (20%) in cancellous bone (FIGS. 13B-C).Further, mice receiving iPTH alone or combined with BT-GSI inducedelevated mRNA levels of Alpl, Runx2, Bglap, and Wnt target genes, andreduced mRNA level of Sost in bone (FIGS. 13 D-E).

These results demonstrate that bone-targeted inhibition of the Notchpathway in the frame of anabolic PTH signaling induces a superior bonegain compared to individual treatments and provide the bases for noveltherapeutic strategies that reduce bone catabolism while simultaneouslypreserve bone anabolism.

While the novel technology has been illustrated and described in detailin the figures and foregoing description, the same is to be consideredas illustrative and not restrictive in character, it being understoodthat only the preferred embodiments have been shown and described andthat all changes and modifications that come within the spirit of thenovel technology are desired to be protected. As well, while the noveltechnology was illustrated using specific examples, theoreticalarguments, accounts, and illustrations, these illustrations and theaccompanying discussion should by no means be interpreted as limitingthe technology. All patents, patent applications, and references totexts, scientific treatises, publications, and the like referenced inthis application are incorporated herein by reference in their entiretyto the extent they are not inconsistent with the explicit teachings ofthis specification.

We claim:
 1. A compound comprising: a compound of the formula A-Y-B;wherein: A is at least one agent that reduces and/or inhibits theactivitiy of gamma-secretase; B is at least one bone-targeting molecule;and Y is a linker that joins and/or links A and B; or a pharmaceuticallyacceptable salt thereof, or a metabolite thereof.
 2. The compound ofclaim 1, wherein: A is the at least one agent that reduces and/orinhibits the activitiy of gamma-secretase; Y is the linker comprisingthe formula NR₁; R₁ is NR₂R₃, NR₂S(=O)₂R₃ or R₂OR₃; R₂ and R₃ areindependently selected from H, C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, C₆H₅OR₄,benzoyl isoleucine, leucine aldehyde, phenyl optionally substituted withC₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, carbonyl, or amide, or benzyl optionallysubstituted with C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, carbonyl, or amide; R₄ isC₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, carbonyl, or amide; and B is at least onebisphosphonate optionally substituted with OH, halogen, CH₃, NH₂,N-alkyl, or N-dialkyl; or a pharmaceutically acceptable salt thereof, ora metabolite thereof.
 3. The compound according to any one of claims1-2, wherein A is a gamma-secretase inhibitor comprising the formula:

.
 4. The compound according to any one of claims 1-2, wherein thecompound comprises one or more stereoisomers of the formula:

wherein: ‘R is H, CH₃, alkyl, halogen, CF₃, CN, OH, OCH₃, or O-alkyl; nis 1-9; m is 0-7; and X is H, OH, halogen, CH₃, NH₂, N-alkyl, orN-dialkyl; or a pharmaceutically acceptable salt thereof, or ametabolite thereof.
 5. The compound according to claim 4, wherein n is1-3, m is 0, and X is H.
 6. The compound according to any of thepreceding claims, wherein said compound is a compound of formula:

or a pharmaceutically acceptable salt thereof, or a metabolite thereof.7. The compound according to according to claim 4, wherein the one ormore stereoisomers comprise any one or more of the formula:

.
 8. A method of reducing the growth of myeloma cells and/or osteoclastdifferentiation, comprising the steps of: administering to a subject atleast one therapeutically effective dose of a compound of any one ofclaims 1-7 or a pharmaceutically acceptable salt or metabolite thereof.9. The method according to claim 8, further comprising the step of:administering to the subject at least one therapeutically effective doseof parathyroid hormone.
 10. The method according to any one of claims8-9, further comprising the step of: administering to the subject atleast one therapeutically effective dose of at least one proteasomeinhibitor.
 11. The method according to claim 10, wherein the at leastone proteasome inhibitor comprises lactacystin, disulfiram,epigallocatechin-3-gallate, marizomib (salinosporamide A), oprozomib(ONX-0912), delanzomib (CEP-18770), epoxomicin, beta-hydroxybeta-methylbutyrate, bortezomib, carfilzomib, and/or ixazomib.
 12. Themethod according to any one of claims 8-11, wherein the subjectcomprises a human, an animal, a cell, and/or a tissue.
 13. A method oftreating a bone related disease, comprising the steps of: administeringto a subject at least one therapeutically effective dose of a compoundof any one of claims 1-7 or a pharmaceutically acceptable salt ormetabolite thereof.
 14. The method according to claim 13, furthercomprising the step of: administering to the subject at least onetherapeutically effective dose of parathyroid hormone.
 15. The methodaccording to any one of claims 13-14, further comprising the step of:administering to the subject at least one therapeutically effective doseof at least one proteasome inhibitor.
 16. The method according to claim15, wherein the at least one proteasome inhibitor comprises lactacystin,disulfiram, epigallocatechin-3-gallate, marizomib (salinosporamide A),oprozomib (ONX-0912), delanzomib (CEP-18770), epoxomicin, beta-hydroxybeta-methylbutyrate, bortezomib, carfilzomib, and/or ixazomib.
 17. Themethod according to any one of claims 13-16, wherein the bone relateddisease comprises osteopenia, osteoporosis, rheumatoid arthritis,hematologic, gastrointestinal and pulmonary disease, autoimmunity,transplant rejection, multiple myeloma, bone cancer, brain cancer,breast cancer, endocrine cancer, gastrointestinal cancer, gynecologiccancer, prostate cancer, head and neck cancer, hematologic cancer, lungcancer, renal cell carcinoma, skin cancer, urologic cancer, and/or rarecancer.
 18. The method according to any one of claims 13-17, wherein thesubject comprises a human, an animal, a cell, and/or a tissue.
 19. Amethod of treating a bone related disease, comprising the steps of:administering to a subject at least one therapeutically effective doseof at least one agent that reduces and/or inhibits the activitiy ofgamma-secretase, or a pharmaceutically acceptable salt or metabolitethereof, and at least one bisphosphonate, or a pharmaceuticallyacceptable salt or metabolite thereof.
 20. The method according to claim19, further comprising the step of: administering to the subject atleast one therapeutically effective dose of parathyroid hormone.
 21. Themethod according to any one of claims 19-20, further comprising the stepof: administering to the subject at least one therapeutically effectivedose of at least one proteasome inhibitor.
 22. The method according toclaim 21, wherein the at least one proteasome inhibitor compriseslactacystin, disulfiram, epigallocatechin-3-gallate, marizomib(salinosporamide A), oprozomib (ONX-0912), delanzomib (CEP-18770),epoxomicin, beta-hydroxy beta-methylbutyrate, bortezomib, carfilzomib,and/or ixazomib.
 23. The method according to any one of claims 19-22,wherein the at least one agent that reduces and/or inhibits theactivitiy of gamma-secretase comprises a compound having the formula:

.
 24. The method according to any one of claims 19-23, wherein the bonerelated disease comprises osteopenia, osteoporosis, rheumatoidarthritis, hematologic, gastrointestinal and pulmonary disease,autoimmunity, transplant rejection, multiple myeloma, bone cancer, braincancer, breast cancer, endocrine cancer, gastrointestinal cancer,gynecologic cancer, prostate cancer, head and neck cancer, hematologiccancer, lung cancer, renal cell carcinoma, skin cancer, urologic cancer,and/or rare cancer.
 25. The method according to any one of claims 19-24,wherein the subject comprises a human, an animal, a cell, and/or atissue.